Cloning Problem

Hi all, I have a recurring problem in cloning a cDNA into a retroviral plasmid vectors, i.e. pMSCV-hCD4.

This is the outline of the procedure:

Both the insert (cDNA) and vector (pMSCV-hCD4) were double-digested with the same set of REs, after which they were ligated and transformed into competent E. coli strain DH5alpha. Upon plating onto an ampicilin-containing agar plate and incubated overnight, colonies were picked and inoculated into both a new ampicilin-containing agar plate (as master plate) and 3 mL of ampicilin-containing broth. Plasmid miniprep was subsequently performed on the overnight liquid cultures. Here is the problem:

For clones with plasmids that electrophoresed at a speed lower than that of the vector, they were confirmed for the presence of an insert by double digestion with the same set of REs used for cloning. Some of these clones had either a larger or a smaller insert, and for those with the desired size, they failed to grow when the corresponding clones on the master plate were inoculated into 100 mL of ampicilin-containing broth for large preparation of the clones (as well as in 3 mL of ampicilin-containing broth). Hence, the plasmid clones, which had the insert of the correct size, were transformed into competent E. coli strain DH5alpha. When colonies were picked from the plate from this step, most of them failed to grow in 3 mL of ampicilin-containing broth and for those that did grow, they had either no plasmid or had one with a size different than that of the desired.

I have repeated this experiment countless times, and the results are consistent. The same cDNA was also cloned into another retroviral vector, pMSCV-GFP, which differs from the former by the marker gene only. Everything went smooth with this latter vector.

you might experience these problemes due to the fact that you might clone sequences toxic to E. coli. It often happens that eukaryotic cDNA contains sequence elements that form cruciform structures in E. coli preventing plasmid replication ...these sequences are then deleted from the plasmid or often sequenc elements get duplicated to stabilize the DNA. Maybe you can do a Z-hunt (http://bioinfo.cgrb.oregonstate.edu/zDNA/) on your insert and look if it is prone to form Z-DNA (unstable in E. coli). If this should be the case you can purchase strains that tolerate such sequences better than others.
If you want to i can provide you with some literature on this topic!

Another issue could be mobile elements that hop into your plasmid ... for example kanamycin is prone to provoke IS1 transposons to hop out of the genome and into plasmids.

you might experience these problemes due to the fact that you might clone sequences toxic to E. coli. It often happens that eukaryotic cDNA contains sequence elements that form cruciform structures in E. coli preventing plasmid replication ...these sequences are then deleted from the plasmid or often sequenc elements get duplicated to stabilize the DNA. Maybe you can do a Z-hunt (http://bioinfo.cgrb....state.edu/zDNA/) on your insert and look if it is prone to form Z-DNA (unstable in E. coli). If this should be the case you can purchase strains that tolerate such sequences better than others. If you want to i can provide you with some literature on this topic!

Another issue could be mobile elements that hop into your plasmid ... for example kanamycin is prone to provoke IS1 transposons to hop out of the genome and into plasmids.

Regards,p

Thanks pDNA! Yea, we initially speculated that the cDNA might be toxic for the E. coli to grow. But if this is the case, how could we explain the fact that when this cDNA is cloned into another plasmid (same plasmid backbone, just different marker gene, i.e. one with GFP, the other with hCD4), the same E. coli carrying this other plasmid can easily be cultured as usual?

Also, I uploaded the plain text file of my insert into the Z-hunt web page as recommended. I was expecting a result that shows if my insert is prone to formation of Z-DNA, but all I got was a percentage breakdown of the nucleotides in this insert. How do we analyse the result? There was no Z-score displayed.

normally, you should get a a Z-score for different sequences in your cDNA ...so the programm would generate a output with different sequence stretches having different Z-scores ...the higher the Z-score the higher the probability that this sequence indeed will form Z-DNA.